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AGROBEST: an Efficient Agrobacterium-Mediated Transient

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AGROBEST: an Efficient Agrobacterium-Mediated Transient Wu et al. Plant Methods 2014, 10:19 http://www.plantmethods.com/content/10/1/19 PLANT METHODS METHODOLOGY Open Access AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings Hung-Yi Wu1,2, Kun-Hsiang Liu3,4, Yi-Chieh Wang1, Jing-Fen Wu1, Wan-Ling Chiu3,4,5, Chao-Ying Chen2, Shu-Hsing Wu1, Jen Sheen3,4 and Erh-Min Lai1,2,3,4* Abstract Background: Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. Results: We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter- gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. Conclusions: AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein–protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer. Keywords: Agrobacterium, Arabidopsis, Transient transformation, Gene expression, Innate immunity, Gain-of-function Background transient gene expression via Agrobacterium-mediated Agrobacterium-mediated DNA transfer is currently the DNA transfer in different plant tissues offers a simple and most facile and versatile method to deliver gene constructs fast method to analyze transgene functions, which is into the nucleus for gene function analysis in diverse plant amenable for high-throughput screens. The transient ex- species [1-3]. Although stable integration of physiologically pression assay is also ideal for systematic dissection of the active and regulated transgenes is the ultimate goal, exquisite and complex processes of Agrobacterium–plant interactions and DNA transfer events [4-7]. * Correspondence: [email protected] Agrobacterium tumefaciens is a soil phytopathogen that 1Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, naturally infects plant wound sites and causes crown gall Taiwan disease via delivery of transferred (T)-DNA from bacterial 2Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 10617, Taiwan cells into host plant cells through a bacterial type IV Full list of author information is available at the end of the article © 2014 Wu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wu et al. Plant Methods 2014, 10:19 Page 2 of 16 http://www.plantmethods.com/content/10/1/19 secretion system (T4SS) [8]. Although Agrobacterium is Pattern-triggered immunity (PTI) induced by a microbe- considered a wound-associated pathogen, it can transfer or pathogen-associated molecular pattern (MAMP or DNA into diverse host cells or tissues under unwounded PAMP) is the first line of active defense in both plants and conditions [9-13]. Interestingly, most of the Arabidopsis animals against pathogens [28-30]. Previous studies have mutants that are resistant to Agrobacterium transformation suggested that Agrobacterium-mediated transformation effi- identified by root explant assays remain highly transform- ciency may be compromised when plants recognize Agro- able by floral dip transformation [14]. The mechanisms and bacterium MAMPs by corresponding pattern-recognition plant factors involved in Agrobacterium-mediated trans- receptors (PRRs) to trigger PTI and block Agrobacterium formation may differ between wounded and unwounded infection [22,24]. The elongation factor Tu (EF-Tu) recep- cells or different tissues. However, the mechanisms under- tor mutant efr-1, which cannot sense EF-Tu MAMP, lying Agrobacterium infection in unwounded cells/tissues showed increased Agrobacterium-mediated transient ex- have not been explored. pression efficiency, as did transgenic Arabidopsis express- In plant biology research, Arabidopsis mesophyll- ing a potent bacterial effector AvrPto to suppress PTI protoplast transfection [15,16] and Agrobacterium-medi- signaling with agroinfiltration of 4- to 5-week-old leaves ated leaf infiltration in Nicotiana benthamiana [17] are [22,24]. However, whether these immune-compromised the well-established and commonly used platforms for Arabidopsis plants are amenable to increase Agrobacterium- transient gene expression analysis. The Arabidopsis mediated transient expression efficiency in young seed- mesophyll-protoplast transient expression system allows lings has not been tested. Defining the condition for for versatile and high-throughput analyses of diverse reliable and highly efficient transformation in healthy gene functions and signal transduction pathways; ad- Col-0 seedlings will be extremely valuable but has never vanced skills with training and practice are essential for been achieved. successful use of this powerful tool for gene function In this study, we systematically investigated various bio- studies [16,18,19]. Agrobacterium-mediated transient ex- logical factors and growth variances to define a combin- pression methods by leaf infiltration have been devel- ation of key factors that contribute to the unprecedentedly oped for a wide range of plants including Nicotiana, high transient transformation and reporter gene expression lettuce, tomato, and Arabidopsis [20-23]. However, the efficiency in Arabidopsis seedlings. As a result of these use of 4- to 5-week-old adult plants with manual infiltra- investigations, we developed an optimized AGROBEST tion has limited application in high-throughput analyses. (Agrobacterium-mediated enhanced seedling transform- Furthermore, although Arabidopsis is the most-studied ation) method that enabled high transient transform- model plant with superbly annotated genome sequences ation and expression efficiency in both efr-1 mutant and and powerful genetic and genomic resources mostly Col-0 Arabidopsis seedlings. Importantly, we demon- available for the Columbia (Col) accession, achieving strated the versatile applicability of AGROBEST in gain- highly efficient and consistent transient expression in of-function studies for the MYB75 transcription factor Col by adult leaf infiltration is challenging [22,24]. in specific target-gene activation and for GIGANTEA (GI) The use of young seedlings for Agrobacterium-mediated reporter gene expression regulated by the Arabidopsis cir- transient expression assays will greatly simplify and cadian clock. The AGROBEST method is a fast, simple, amplify the power of the method. Indeed, Agrobacterium- reliable, and versatile tool for systematic gene function mediated transient expression in Arabidopsis seedlings analysis and a new tool for dissecting the Agrobacterium- has been recently developed for fast and robust analysis mediated DNA transfer processes. of protein subcellular localization and protein–protein interactions [25-27]. The system’s requirement for high- Results density Agrobacterium cells and vacuum infiltration [27] Cotyledons of young Arabidopsis EF-TU receptor mutant or chemical treatment (e.g., the addition of surfactant is highly susceptible to Agrobacterium-mediated transient Silwet L-77) [26] to achieve high cellular transformation transformation efficiency could induce innate immunity and stress re- Environmental and biological factors such as growth sponses in plants, which globally alters cellular, physio- conditions, host plants, and Agrobacterium strains can logical, and signaling processes and severely retards affect the transformation efficiency. We first evaluated growth [28,29]. Thus, developing a system that circum- the
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